A purine metabolic checkpoint that prevents autoimmunity and autoinflammation

Summary Still’s disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still’s disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation.


Correspondence
In brief Saveljeva et al. identify a biochemical mechanism in dendritic cells that restrains T cell priming and prevents immunopathology but dampens tumor surveillance. FAMIN enables a purine nucleotide cycle, which prevents cytoplasmic NADH/NAD + reductive stress that augments antigen presentation, and it generates inosine, which inhibits T cell activation.

INTRODUCTION
Deorphaning an autoimmunity risk gene product unearthed an unprecedented function at the heart of cellular metabolism, conserved from bacteria to man. This risk gene encodes FAMIN (also known as LACC1, C13orf31), an enzyme that unifies in a single protein the activities of adenosine deaminase (ADA; adenosine + H 2 O . inosine + NH 3 ), purine nucleoside phosphorylase (PNP; inosine + phosphate [P i ] # hypoxanthine + ribose-1-phosphate [R1P]; guanosine + P i # guanine + R1P), and methylthioadenosine phosphorylase (MTAP; methylthioadenosine [MTA] + P i # adenine + methyl-thioribose-1-phosphate [MTR1P]). FAMIN's fourth catalytic activity is that of an adenosine phosphorylase (adenosine + P i # adenine + R1P), previously considered absent from eukaryotic metabolism (Cader et al., 2020). Adenine and ribose are primordial metabolites from which life is thought to have emerged from prebiotic biochemistry (Ralser, 2018). They are defining constituents of the genetic code, the energy currency, and the major cofactors of a cell. Since purine nucleotide de novo synthesis yields straight to nucleotides (i.e., purine monophosphates; IMP, AMP, and GMP), ADA, PNP, and MTAP had been thought to be the sole enzymes to supply purine nucleobases (adenine, guanine, and hypoxanthine) from nucleosides (adenosine, guanosine, inosine, and MTA) (Bzowska et al., 2000). ADA and PNP deficiency causes severe combined immunodeficiency (SCID) with loss of T and B lymphocytes (Giblett et al., 1972(Giblett et al., , 1975. Loss of function of FAMIN, in sharp contrast, is linked to autoimmunity and autoinflammation, specifically to Still's disease (also known as systemic juvenile idiopathic arthritis, sJIA), juvenile idiopathic arthritis (JIA), and early-onset Crohn's disease (Al-Mayouf et al., 2020;Patel et al., 2014;Rabionet et al., 2019;Wakil et al., 2015;Yasin and Schulert, 2018). These very rare loss-of-function mutations aside, partial loss of activity, caused by a SNP that leads to a valine-for-isoleucine substitution at amino acid 254 (I254V), for which $6% of humans are homozygous, increases risk of Crohn's disease and leprosy (Barrett et al., 2008;Zhang et al., 2009). FAMIN loss of function is the sole known cause of autosomalrecessive (i.e., monogenic) forms of Still's disease. Still's disease affects young children; starts with daily recurring fever, rash, and lymph node enlargement; and morphs over weeks into a debilitating arthritis (Yasin and Schulert, 2018). The initial phase resembles periodic fever syndromes with inflammasome activation and IL-1b and IL-18 release; the later arthritic phase is thought to be driven by pathogenic T lymphocytes. About 20% of children with Still's disease develop ''secondary hemophagocytic lymphohistiocytosis'' (HLH; also known as ''macrophage activation syndrome,'' MAS) (Brisse et al., 2016b;Grom et al., 2016). HLH/MAS is typically triggered by viral infections and can occur even when Still's disease is in remission while on IL-1/IL-6-blocking therapeutics (Grom et al., 2016). HLH features a cytokine storm accompanied by excessive expansion and activation of CD4 + and CD8 + T lymphocytes and hemophagocytic, IFNg-activated macrophages. It manifests with disseminated intravascular coagulation (DIC), acute respiratory distress syndrome, and multi-organ failure, and is often fatal (Brisse et al., 2016a(Brisse et al., , 2016b. HLH/MAS is not restricted to Still's disease and children. For example, virus-induced HLH/MAS is caused by Epstein-Barr virus and many other pathogens and has been implicated in fatality from seasonal (H3N2), avian (H5N1), and swine (H1N1/2009) influenza A virus (IAV) infections (Beutel et al., 2011;Henter et al., 2010).
Mice with germline deletion of Famin, or genome-edited to express one of the Still's disease-linked loss-of-function mutations (C284R; ''Famin p.284R '' mice), develop normally under specific pathogen-free conditions. Similarly, mice genome-edited to express fully active (254I; ''Famin p.254I '') or partially active FAMIN (254V; ''Famin p.254V '') are indistinguishable . Famin À/À and Famin p.284R mice, however, do develop more severe lipopolysaccharide (LPS)-induced sepsis, evidence of DIC, and increased plasma IL-1b levels, compared to mice expressing fully active FAMIN . Compromised FAMIN activity also leads to lower reactive oxygen species (ROS) production, decreased bacterial killing, altered NLRP3 inflammasome activation, and cytokine secretion in macrophages Lahiri et al., 2017), and Famin À/À mice develop more severe experimental arthritis and colitis Skon-Hegg et al., 2019). How loss of FAMIN activity, which is abundantly expressed in macrophages and dendritic cells (DCs) while largely absent from T cells (Heng et al., 2008), predisposes for autoimmunity remains unknown. Particularly elusive is via what mechanism altered core purine metabolism due to the absence of multifunctional FAMIN, which is tethered to the cytoplasmic surface of peroxisomes , could affect immune function, since monofunctional ADA, PNP, and MTAP are ubiquitously present.
Here we report a purine metabolon in DCs that potently restrains T cell priming by dampening membrane trafficking and hence the pace of antigen uptake and presentation, and by releasing inosine that provides an inhibitory signal via the adenosine A 2A receptor (A 2A R). Impaired FAMIN catalysis results in excessive IAV-specific T cell responses and lung immunopa-thology, but also in enhanced tumor immune surveillance. We describe a purely biochemical mechanism within DCs that exerts fundamental control over T lymphocyte priming.

FAMIN in DCs restrains priming of class I and class II-restricted antigens
Intrigued by the selective increase in T cell responses emanating from FAMIN deficiency in DCs, we focused our study on whether and how FAMIN controls T cell priming and turned to ovalbumin (OVA) as a model antigen. Baseline percentages of splenic and lymph node CD4 + and CD8 + T lymphocytes, and splenic cDC1s and cDC2s, were indistinguishable between Famin p.254I , Famin p.254V , and Famin p.284R mice (Table S1). Splenic CD11c + DCs from Famin DDC mice primed naive OVA 257-264 -specific OT-I T lymphocytes for increased expansion and IFNg secretion compared to those primed by Famin WT DCs, irrespective of whether they were pulsed with OVA 257-264 peptide, OVA protein, or necrotic fibroblasts expressing a non-secreted OVA (bm1T-OVA; Sancho et al., 2009) requiring cross-presentation ( Figures  2A-2C). Antigen-specific cytotoxicity, IFNg, and granzyme B release were higher when naive OT-I T cells had been primed by Famin DDC than by Famin WT splenic DCs ( Figures 2D and 2E). CD8a + conventional DCs type 1 (cDC1) preferentially prime naive CD8 + T cells, and CD11b + cDC2 preferentially CD4 + T cells (Durai and Murphy, 2016). Exaggerated cytotoxic T lymphocyte (CTL) responses were similarly observed when primed by bone marrow (BM)-derived Famin À/À compared to Famin +/+ cDC1 ( Figure S2A), hence extending to DCs immunologically distinct from splenic DCs (Naik et al., 2005). BM-derived cDC1 from Famin p.284R and Famin À/À mice also primed OT-I T cells for higher proliferation and IFNg secretion compared to those from Famin p.254I and Famin p.254V mice ( Figure S2B). Restimulation of cDC1-OT-I T cell co-cultures after 72 h ( Figure 2F), or after further differentiation over 6 days into antigen-specific T effector (T E ) and T effector memory (T EM ) cells via IL-2 and IL-15 (Figure 2G), respectively, resulted in highest IFNg secretion when priming was provided by Famin p.284R and Famin À/À cDC1, inter-mediate by Famin p.254V , and lowest by Famin p.254I cDC1. Hence, CD8 + T cell responses increased with decreasing FAMIN activity in DCs, and the enhanced priming effect persisted when further differentiated into T E and T EM cells.
To investigate this in vivo, OT-I T cells were adoptively transferred into Famin DDC and Famin WT mice followed by intraperitoneal immunization with ovalbumin, and CTL activity was assessed 4 days later. OVA 257-264 -specific cytotoxicity, IFNg, and granzyme B release of splenic T cells were strikingly higher in Famin DDC compared to Famin WT mice ( Figure 2H). Increased CTL activity was intrinsic, as OT-I proliferation in vivo was similar between genotypes upon adjuvant-free priming ( Figure S2C). Hence, lack of FAMIN activity in DCs enhanced their ability to prime CD8 + T cell responses to a model antigen in vivo.
FAMIN controls DC metabolism and tunes antigen uptake and presentation without a transcriptional signature Despite the profound differences in their priming activity, Famin itself and genomically adjacent Ccdc122 were the sole differentially expressed genes (DEGs) in Famin À/À compared to Famin +/+ BM-derived cDC1 analyzed by RNA sequencing (RNA-seq; Figure 3A). A comparison of cDC1 from Famin p.254I and Famin p.254V mice did not reveal a single DEG ( Figure S3A), and only 56 upand 32 downregulated transcripts between Famin p.284R and Famin p.254I cDC1 ( Figure S3B; Table S2). Among those DEGs were only four (Fcgr1, Tlr7, Ikbkg, and Lcn2) encoding immune mediators, and no enrichment for gene ontology processes linked to DC activation was observed (data not shown). Famin genotype did not affect protein expression of ADA, PNP, and MTAP ( Figure S3C), which share catalytic activities with FAMIN. Liquid chromatography-mass spectrometry (LC-MS) demonstrated a marked reduction in purine nucleotides from Famin p.254I via Famin p.254V to Famin p.284R cDC1 (Figures 3B  and 3C; Table S3), raising the possibility of a bona fide biochemical mechanism controlling T cell priming.
Priming of naive T cells entails T cell receptor (TCR) binding to a peptide-MHC complex on a professional antigen-presenting cell, which is then fine-tuned by costimulatory molecules and secreted mediators (Blum et al., 2013;Cantrell, 2015). A pulse with a fluorescent AF647-ovalbumin conjugate (AF647-OVA) as model antigen demonstrated higher uptake, particularly at earlier time points, in Famin p.284R compared to Famin p.254I splenic DCs, with intermediate levels in Famin p.254V cells ( Figure 3D). Higher AF647-OVA uptake in Famin p.284R and Famin p.254V compared to Famin p.254I genotypes was observed in both cDC1 and cDC2 splenic DCs, as well as in BM-derived cDCs ( Figures  S3D and S3E). This suggested that in the absence of FAMIN activity, antigen uptake was accelerated and available for presentation via MHC class II for CD4 + T cells and cross-presentation via MHC class I for CD8 + T cells, the latter requiring endosome-to-cytosol transfer (Blander, 2018). This can be measured using endocytosed b-lactamase in DCs that are pre-loaded with a cytosolic probe that loses its FRET signal upon b-lactamase cleavage, when the latter gains access to the cytosol . Compared to Famin p.254I cells, Famin p.284R splenic DCs exhibited increased probe cleavage, especially at the earliest time point, demonstrating increased endosome-tocytosol transfer ( Figure 3E). The peptide repertoire presented on surface MHC I is continuously optimized by peptide exchange in the endoplasmic reticulum (ER) (Williams et al., 2002). Increased staining with monoclonal antibody 25-D1.16, which recognizes OVA 257-264 bound to H-2K b (Porgador et al., 1997), directly demonstrated increased peptide presentation on Famin p.284R and Famin p.254V compared to Famin p.254I splenic DCs after a pulse with OVA 257-264 ( Figures 3F and S3F). The difference between Famin genotypes in peptide:MHC I complexes was again most pronounced early after the OVA 257-264 pulse, indicative of FAMIN controlling the pace of the process. Famin genotype did not affect total surface MHC I and II expression (Table S4; Figure S3G). Hence, loss of FAMIN activity led to faster-paced endosomal antigen uptake, transfer to cytosol, and peptide exchange and presentation on MHC I. FAMIN can promote flux through a cycle that interconverts IMP to succinyl-AMP (S-AMP), AMP, and back to IMP via sequential activities of adenylosuccinate synthase (ADSS), adenylosuccinate lyase (ADSL), and AMP deaminase (AMPD) ( Figure 3G) (Cader et al., 2020). In skeletal muscle and macrophages, the IMP-S-AMP-AMP cycle promotes energy metabolism and is referred to as purine nucleotide cycle (PNC) (Cader et al., 2020;Lowenstein and Tornheim, 1971). Cellular levels of IMP, S-AMP, and AMP decreased from Famin p.254I and Famin p.254V to Famin p.284R BMderived cDC1 ( Figures 3B and S3H). Tracing [ 13 C 16 ] palmitate, we observed decreased flux into Krebs cycle metabolites a-ketoglutarate, succinate, and malate in Famin p.254V and Famin p.284R compared to Famin p.254I BM-derived DC1s ( Figure S3I). We also detected decreased onward flux into aspartate (which enters the IMP-S-AMP-AMP cycle) in Famin p.284R DCs. A similar pattern (D and E) Specific cytotoxicity against OVA 257-264 -pulsed wild-type splenocytes (D), and IFNg and granzyme B release (E) of OT-I T cells that had been primed with Famin WT or Famin DDC splenic DCs pulsed with OVA 257-264 (n = 3). (F) IFNg secretion from re-stimulated (OVA 257-264 for 5 h) OT-I T cells after 72 h of priming with Famin p.254I , Famin p.254V , Famin p.284R , or Famin À/À BM-derived cDC1 pulsed with OVA 257-264 (n = 3). (G) IFNg secretion after 5 h anti-CD3/CD28 re-stimulation of OT-I T cells that had been differentiated into T E and T EM cells, following 72 h of priming by Famin p.254I , Famin p.254V , Famin p.284R , and Famin À/À BM-derived cDC1 pulsed with OVA 257-264 (n = 3). (H) OVA 257-264 -specific cytotoxicity, granzyme B, and IFNg secretion of splenocytes of Famin DDC and Famin WT mice that had been adoptively transferred with naive OT-I T cells and immunized with ovalbumin 72 h earlier (n = 3). (I) Proliferation indices of OT-II T cells 96 h after priming with OVA 323-339 -pulsed splenic Famin +/+ and Famin À/À DCs (n = 3). (J and K) IFNg (J) and IL-2 (K) in supernatants of OT-II cells 96 h after priming with OVA 323-339 -pulsed splenic Famin +/+ and Famin À/À DCs (n = 3). (L) Percentage IFNg + OT-II T cells after restimulation with anti-CD3/CD28, following priming with OVA 323-339 -pulsed BM-derived cDC2 7 days earlier (n = 3). (M) Proliferation indices of OT-II T cells adoptively transferred into Famin DDC and Famin WT mice 72 h after immunization with ovalbumin (n = 3). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA or unpaired two-tailed Student's t test where appropriate). See also Figure S2.   Figure 3. FAMIN controls DC metabolism and tunes antigen uptake and presentation without a transcriptional signature (A) Differentially expressed genes between Famin À/À and Famin +/+ BM-derived cDC1s (n = 4; GEO: GSE126473).
(legend continued on next page)  Figure S3I). In contrast, flux from [ 13 C 5 15 N 2 ] glutamine into Krebs cycle metabolites and aspartate increased in FAMIN-impaired cDC1s ( Figure S3I). Overall, this was consistent with perturbed fatty acid oxidation (FAO) and lipid carbon channeling into the PNC, as well as compensatory changes in glutamine metabolism, mirroring key observations in FAMIN-deficient macrophages (Cader et al., 2020). Consequently, cDC1s' oxygen consumption rate (OCR), reflecting oxidative phosphorylation (OXPHOS), decreased from Famin p.254I via Famin p.254V to Famin p.284R genotypes ( Figure 3H). The extracellular acidification rate (ECAR) was also lower in Famin p.284R compared to Famin p.254I BM-derived cDC1s ( Figure 3I), and secretion of lactate, with which protons (H + ) are co-exported, correspondingly declined from Famin p.254I to Famin p.254V and Famin p.284R DCs ( Figure 3J). Corresponding observations were made in splenic DCs ( Figure S3J), in which cDC2 predominate over cDC1 (Table S1). The cytoplasmic pH (pH c ) of cDC1 (data not shown) and splenic DCs became more acidic as FAMIN activity decreased ( Figure 3K). This demonstrated that FAMIN promotes DCs' energy metabolism and prevents cytoplasmic acidification. By consuming aspartate and releasing its carbons as fumarate, which can be hydrated to malate, the IMP-S-AMP-AMP cycle can affect electron (e À ) transfer between cytoplasm and mitochondria, which ensues via the malate-aspartate shuttle (Borst, 2020;Cader et al., 2020). The aspartate pool supplying the IMP-S-AMP-AMP cycle was inaccessible by exogenously supplied [ 13 C 4 15 N 1 ] aspartate ( Figure 3L), similar to most cells in culture (Birsoy et al., 2015). Fractional incorporation of exogenously provided [ 13 C 4 ] malate was strikingly higher in Famin p.284R compared to Famin p.254I and Famin p.254V BM-derived DCs, as levels of unlabeled malate were conversely lowest in Famin p.284R and highest in Famin p.254I cells ( Figures 3M and S3K). These differences in cellular malate, encompassing cytoplasmic and mito-chondrial pools, corroborated that energy metabolism is pervasively perturbed in FAMIN-impaired DCs. Exogenous malate resulted in marked differences in levels of aspartate, IMP, S-AMP, and AMP between Famin genotypes ( Figure 3N). Altogether this pointed, in analogy to macrophages (Cader et al., 2020), to the IMP-S-AMP-AMP cycle as an immediate biochemical effector of FAMIN catalysis.
IMPDH-dependent NADH/NAD + redox state controls the pace of antigen uptake and MHC I recycling Bypassing IMPDH and GMPS with exogenous guanine increased AF647-OVA uptake in Famin p.254I and Famin p.254V splenic DCs, phenocopying enhanced antigen uptake of Famin p.284R DCs (Figure 5A). In the latter, guanine did not further augment uptake (Figure 5A). Prima vista, this suggested that guanine nucleotide pools may control antigen uptake. Increased antigen uptake, however, was at odds with decreased GTP and GDP levels in FAMINimpaired DCs ( Figure 3B). This implied a byproduct of the IMP-XMP-GMP cycle, rather than guanine nucleotide pool size, may be responsible for increased membrane trafficking. IMPDH reduces NAD + to NADH + H + ( Figure 3G). Inhibition of IMPDH rescued cytoplasmic acidification in Famin p.284R DCs ( Figure 5B), adding further evidence for enhanced flux through IMPDH. An altered pH c can affect vesicular trafficking (Heuser, 1989;Korolchuk et al., 2011;Walton et al., 2018). ADSS inhibition in Famin p.254I DCs enhanced AF647-OVA uptake ( Figure 4A) without causing cytoplasmic acidification ( Figure 5C), arguing against pH c changes accounting for altered membrane trafficking. IMPDH inhibition did not rescue OCR or ECAR deficits in Famin p.284R DCs ( Figures 5D and 5E), confirming that compromised OXPHOS and glycolysis are not directly responsible for exaggerated antigen uptake. As total cellular NAD(H) integrates protein-bound and free forms across cytoplasmic and mitochondrial pools with their distinct redox states, we measured the secreted lactate/pyruvate ratio to deduce the cytosolic free NADH/NAD + ratio (Goodman et al., 2020;Krebs, 1967;Williamson et al., 1967). Famin p.284R splenic DCs exhibited a markedly higher lactate/pyruvate ratio than Famin p.254I cells ( Figure 5F), implying increased cytosolic NADH/NAD + . In contrast, the secreted b-hydroxybutyrate/acetoacetate ratio, reflecting mitochondrial free NADH/ NAD + , remained unchanged ( Figure S5A). To investigate whether re-oxidation of cytoplasmic NADH rescues exaggerated antigen uptake, we provided pyruvate as external e À acceptor that regenerates NAD + via lactate dehydrogenase (LDH) ( Figure 5G). Pyruvate indeed rescued increased AF647-OVA uptake in Famin p.284R splenic DCs ( Figure 5H). Four-carbon a-ketobutyrate (AKB) is an alternative substrate for regenerating NAD + from NADH via LDH ( Figure 5G) (Sullivan et al., 2015). AKB is primarily used as e À acceptor and not as carbon substrate in other (E and F) Proliferation indices, CFSE overlays (E), and IFNg released (F) from OT-I T cells co-cultured for 72 h with Famin p.254I splenic DCs pulsed with ovalbumin 48 h after nucleofection with ctrl or Adsl, Adss, and Ampd2/Ampd3 siRNAs (n = 6, 3 mice per siRNA).
(G) Schematic depicting the lactate dehydrogenase (LDH) reaction, in which pyruvate is converted to lactate with regeneration of NAD + ; a-ketobutyrate acts as an alternative electron acceptor and is converted to a-hydroxybutyrate.
(H) Percentage AF647-OVA + splenic DCs of indicated genotypes following incubation with pyruvate or a-ketobutyrate (AKB) overnight and replenished for the time of the assay (n = 6, 3 mice per genotype).
(K) Percentage of MHC I recycled in Famin p.284R BM-derived cDC1s at indicated times following overnight incubation with AKB or control (n = 3-5, 3 mice per genotype). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA or unpaired two-tailed Student's t test where appropriate). See also Figure S5. (legend continued on next page) ll OPEN ACCESS Article metabolic pathways. AKB precisely phenocopied the rescue of antigen uptake achieved by pyruvate, decreasing AF647-OVA uptake to equally low levels in Famin p.254I and Famin p.284R splenic DCs ( Figure 5H). AKB reduced the secreted lactate/pyruvate ratio as expected ( Figure S5B), while the pH c difference was retained between Famin p.284R and Famin p.254I splenic DCs ( Figure S5C), consistent with cytoplasmic acidification not accounting for increased antigen uptake. Augmentation of baseline OCR was markedly different between pyruvate and AKB ( Figures S5D and  S5E). This was consistent with only pyruvate entering mitochondrial oxidation ( Figure 5G), which we confirmed by tracing [ 13 C 3 ] pyruvate and [ 13 C 4 ] AKB into Krebs cycle metabolites in Famin p.254I and Famin p.284R cDC1s ( Figures S5F-S5I). Importantly, pyruvate and AKB also rescued increased AF647-OVA uptake in Famin p.254I splenic DCs, in which the IMP-S-AMP-AMP cycle was halted by Cpd3 ( Figure 5I). AF647-OVA uptake serves as proxy of one specialized endocytic pathway, but membrane trafficking is involved in all the different routes of antigen uptake, processing, and (cross-)presentation (Alloatti et al., 2016;Blander, 2018). Considering whether FAMIN-mediated redox control of membrane trafficking is a more general principle, we asked whether FAMIN affects MHC I recycling, required for loading of cross-presented peptides Joffre et al., 2012). MHC I recycling was higher in Famin p.284R compared to Famin p.254I cDC1 ( Figure 5J). AKB markedly reduced MHC I recycling in Famin p.284R , and barely in Famin p.254I cDC1 ( Figures 5K  and S5J), revealing that cytoplasmic NADH/NAD + affects the pace of MHC I recycling, too. This provided strong evidence that enhanced antigen uptake and presentation in FAMINimpaired DCs is caused by increased reduction of cytoplasmic NAD + to NADH by IMPDH due to an imbalance in adenine-guanine nucleotide interconversion cycles.
The FAMIN product inosine dampens T cell activation during priming Fixing splenic DCs with glutaraldehyde after pulsing with OVA 257-264 retained the ability of Famin p.284R compared to Famin p.254I DCs to prime for increased OT-I T cell proliferation ( Figure S6A), but the capacity to prime for enhanced IFNg secretion by Famin p.284R DCs, however, was lost ( Figure S6B). This indicated that optimal priming requires mutual dynamic transmembrane signaling. It also raised the possibility that soluble factors released from DCs might be involved, too. Famin À/À and Famin +/+ DCs, and Famin p.254I , Famin p.254V , and Famin p.284R cDC1s, were indistinguishable in their expression of co-stimulatory and co-inhibitory molecules (Table S4; Figure S6C). Cell-free supernatants of Famin À/À DCs primed naive anti-CD3/CD28activated OT-I T cells to secrete 2-fold more IFNg compared to supernatants of Famin +/+ DCs ( Figure 6A). Transcriptomes of OT-I T cells activated in the presence of Famin À/À compared to Famin +/+ DC supernatants were enriched for hallmark gene sets (Leone et al., 2019) indicative of elevated effector function ( Figures 6B, 6C, S6D, and S6E; Table S6). IL-12p70 and IFNa secretion by Famin À/À and Famin +/+ DCs in co-culture with naive OT-I T cells was indistinguishable ( Figure S6F). Freeze-thaw cycles did not affect Famin À/À DC supernatants' enhanced stimulatory capacity (data not shown), which was retained after passing through a 3 kDa filter ( Figure 6D), pointing to a small molecule. To enable its identification, we switched to serumfree OptiMEM media to reduce complexity. OptiMEM supernatants of Famin À/À and Famin +/+ DCs retained differences in IFNg induction in anti-CD3/CD28-activated CD8 + T cells (Figure S6G). They were particularly stark between those elicited by Famin p.254I compared to Famin p.254V DC supernatants (Figure 6E). CD4 + OT-II T cells were also primed for heightened IFNg secretion by Famin p.254V and Famin p.284R compared to Famin p.254I DC supernatants ( Figure 6F). This suggested that DCs secrete a small molecule in a FAMIN-dependent manner that inhibits priming of naive CD4 + and CD8 + T cells. Unbiased high-resolution LC-MS of supernatants of Famin +/+ and Famin À/À splenic DCs resolved $1,100 features, revealing inosine as the top-ranking identifiable LC-MS feature of differential abundance (Figures 6G and 6H). A second unbiased LC-MS screen comparing Famin p.254I with Famin p.284R splenic (D) IFNg secretion from OT-I T cells activated by anti-CD3/CD28 for 72 h in the presence of <3 kDa cut-off filtrates of supernatants from Famin +/+ or Famin À/À splenic DCs cultured for 3 h in RPMI-1640/10% FBS (n = 3).  Figure S6H) (n = 3-6, from 3 mice per genotype). (P and Q) IFNg secretion (P) and proliferation indices (Q) of OT-I T cells activated by anti-CD3/CD28 for 72 h in the presence of CGS21680 or SCH58261 and supernatants from Famin p.254I , Famin p.254V , and Famin p.284R splenic DCs cultured for 3 h in OptiMEM (n = 6, 3 mice per genotype). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA or unpaired two-tailed Student's t test where appropriate). See also Figure S6.   Article DC supernatants identified two metabolites, inosine and propionyl-carnitine, overlapping with the first screen ( Figure 6I). As a catalytic product of FAMIN, inosine, whose levels were highest with fully active FAMIN-254I ( Figures 6J and 6K), was a plausible candidate. Pure inosine dose-dependently reduced OT-I T cell IFNg secretion and proliferation after anti-CD3/CD28 stimulation ( Figures 6L and 6M). Spiking co-cultures of OVA-presenting splenic DCs and naive OT-I T cells with just 5 nM inosine, the lower end of differentials in inosine levels between Famin p.254I and Famin p.284R DC supernatants ( Figure S6H), was sufficient to decrease T cell proliferation and IFNg secretion ( Figures 6N  and 6O). This suggested inosine as an inhibitory signal during T cell priming. Among the four adenosine receptors, direct binding of, and activation by, inosine has been shown for A 2A R (Welihinda et al., 2018), the only adenosine receptor expressed on T cells (Cekic et al., 2013). In OT-I T cells activated by anti-CD3/CD28 in the presence of DC supernatants, the A 2A R agonist CGS21680 (Hutchison et al., 1989) inhibited proliferation and IFNg secretion and abrogated Famin genotype-related differences ( Figures 6P and 6Q). A 2A R antagonism with SCH58261 (Zocchi et al., 1996) had the converse effect ( Figures 6P and  6Q). This demonstrated that FAMIN-dependent release of inosine from DCs inhibited activation of naive T cells.

FAMIN-dependent conversion of extracellular hypoxanthine into inosine
Nucleobases and nucleosides equilibrate across the plasma membrane via purine/pyrimidine transporters (Boswell-Casteel and Hays, 2017), prompting us to consider whether external nucleobases may supply the substrate for the synthesis of inosine in DCs ( Figure 7A). A 3 h pulse of splenic DCs with 25 mM [ 13 C 5 15 N 4 ] hypoxanthine labeled over half of extracellular and cellular hypoxanthine (Figures S7A-S7D). Labeled hypoxanthine was converted to cellular [ 13 C 5 15 N 4 ] inosine, which was highest in Famin p.254I and lowest in Famin p.284R DCs (Figures 7B and  S7E). This resulted in [ 13 C 5 15 N 4 ] inosine released into supernatants, whose levels were, in relative terms, $35% and $22% higher in Famin p.254I and Famin p.254V , respectively, than in Famin p.284R DCs ( Figure 7C). Even fractional [ 13 C 5 15 N 4 ] inosine labeling increased from Famin p.284R to Famin p.254V and Famin p.254I supernatants, averaging at one-fifth of total (Figure 7C), despite unlabeled inosine levels increased alongside, too ( Figure 7D). Since OptiMEM is inosine-free and levels in RPMI-1640/10% FBS negligible (<100 pM, below the lowest detectable standard; Figure 7E), media change for labeling studies prompt an immediate equilibrative efflux; hence, these marked differences likely underestimate the contribution of FAMIN to inosine release during priming in situ. Plasma levels of inosine were similar across Famin germline mutants and DC-selective deletion (Figures S7F and S7G), consistent with a model of localized release. Perturbation in adenine-guanine nucleotide interconversion revealed increased inosine release upon IMPDH inhibition and a slight decrease upon ADSS and AMPD inhibition, with differential release across Famin genotypes remaining intact (Figures 7F-7H). Altogether these studies demonstrated that elevated inosine release by DCs with active FAMIN amplifies an inhibitory signal during T cell priming, generated by phosphoribosylation of largely extracellularly derived hypoxanthine.

Compromised FAMIN catalysis enhances tumor immune surveillance
We finally turned to tumor immunosurveillance, a model not confounded by increased viral replication associated with IMPDH activity , to assess endogenous CTL function primed in the setting of polymorphic Famin variants. Increased DC antigen presentation enhances tumor-specific immunity by inducing Th1 and CTL responses (Xia et al., 2018). A 2A R signaling prevents T cell anti-tumor immunity by inhibiting CTL activation and maintaining naive T cells quiescent (Cekic et al., 2013;Ohta et al., 2006). Famin p.254I and Famin p.254V mice developed markedly larger tumors compared to Famin p.284R mice ( Figures 7I and 7J) when subcutaneously injected with a syngeneic Lewis lung carcinoma cell line expressing ovalbumin (LL/2-OVA) (Kraman et al., 2010). Protection was associated with nominally higher OVA 257-264 -specific CD8 + T cell numbers in peripheral blood in Famin p.284R compared to Famin p.254I and Famin p.254V mice ( Figure S7H). These results were consistent with augmented priming that translated into increased CTL activity and tumor immunosurveillance when FAMIN activity is compromised.

DISCUSSION
Here we discovered that purine nucleotide and nucleoside turnover in DCs represses T cell immunity, with FAMIN acting as a purely biochemical immune checkpoint. FAMIN achieves this via two main routes. First, FAMIN restrains endocytosis, antigen processing, and presentation via cytoplasmic NADH/NAD + through balancing adenine-guanine nucleotide interconversion. Second, it amplifies an inhibitory signal through the generation of locally released inosine. The relative contribution of altered (C) [ 13 C 5 15 N 4 ] inosine, and its fractional incorporation, in supernatants of Famin p.254I , Famin p.254V , and Famin p.284R splenic DCs pre-equilibrated in OptiMEM for 3 h before a 3 h pulse with [ 13 C 5 15 N 4 ] hypoxanthine in OptiMEM (n = 18, 3 mice per genotype). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA or unpaired two-tailed Student's t test where appropriate). See also Figure S7.

OPEN ACCESS
Article antigen presentation versus altered inosine release to T cell priming is impossible to disentangle, since both are directly catalytically controlled by FAMIN. ADA, PNP, and MTAP do not compensate for FAMIN's absence, although they share three of four of FAMIN's catalytic activities. This suggests that FAMIN is at the center of a dedicated purine metabolon that biochemically restrains DCs' priming activity. Pacing membrane trafficking via adenine-guanine nucleotide interconversion cycles through an NADH/NAD + -sensitive mechanism, consequent to hyperactive IMPDH that reduces NAD + to NADH, may represent a general principle. Vectorial physical membrane displacements, which can be energized by transmembrane e À transport, occur upon plasma membrane internalization and, in the opposite direction, during membrane budding and vesicle formation (Morré and Morré , 2011). NADH can activate vectorial membrane transfer, elegantly demonstrated in cell-free systems for the transfer from the trans Golgi apparatus to the plasma membrane (Rodriguez et al., 1992). Aside from IMPDH, NAD + is reduced to NADH by several cytoplasmic reactions, foremost by glyceraldehyde dehydrogenase of glycolysis, and re-oxidized by LDH and the malate-aspartate shuttle via mitochondria. NADH/ NAD + reductive stress due to perturbation in any of those reactions might therefore also impact membrane trafficking and antigen presentation. NADH/NAD + reductive stress in the liver emerges as the causal mechanism for features of the metabolic syndrome associated with hypomorphic GCKR, such as hepatic insulin resistance and increased triglyceride release (Goodman et al., 2020). GCKR localizes at the probably most pleiotropic genome-wide association study (GWAS) locus and encodes liver-specific glucokinase regulatory protein, which helps prevent a futile metabolic cycle with glycolysis during gluconeogenesis (Goodman et al., 2020). Whether obesity, which increases risk for autoimmunity (Versini et al., 2014), causes NADH/NAD + reductive stress in DCs is unknown.
Via equilibration of purine nucleobases and nucleosides across the plasma membrane, DCs may survey their vicinity. They respond to hypoxanthine by converting it to inosine, dampening T cell activation. Since phosphorolysis is reversible, DCs might bidirectionally respond to local hypoxanthine and inosine availability during immunological synapse formation. T cells, in particular naive CD4 + T cells, can release hypoxanthine (Fan et al., 2019), which may enable a dynamic interaction with FAMIN catalysis in DCs affecting the priming threshold. Systemic hypoxanthine and inosine levels closely track each other, consistent with their rapid interconversion via PNP (Sun et al., 2019). The intestinal microbiota may also affect inosine plasma levels (Mager et al., 2020). Inosine activation of the A 2A R on T cells is itself complex: inosine either prevented Th1 differentiation and blunted anti-tumor immunity in anti-CTLA4-treated mice or, conversely, enhanced both, when co-supplied with IFNg in vitro and a TLR9 agonist in vivo (Mager et al., 2020). The mechanism underlying this switch remained unclear. Inosine can also serve as an alternative carbon source for CD8 + T cells when glucose is unavailable (Wang et al., 2020). The complete (254I versus 254V) and almost-complete (254I versus 284R and Famin +/+ versus Famin À/À ) absence of transcriptomic changes excludes that autocrine inosine-triggered receptor signaling in DCs dampens their priming capacity.
A final point is that experiments in wild-type mice may grossly over-estimate anti-viral and anti-tumor T cell immunity that can be expected in the majority of humans, since mice naturally express hypomorphic FAMIN-254V. As we show here, the single amino acid change to À254I (for which $94% of humans are homozygous or heterozygous) results in 4-fold lower numbers of nucleoprotein-specific CTLs upon experimental IAV infection and a profound reduction in T cell effector function. This polymorphism (rs3764147) has been linked to a possible founder effect (Rivas et al., 2018), hinting that excessive priming may have afforded evolutionary benefits.

Limitations of study
The IMP-S-AMP-AMP cycle operates at the center of energy metabolism, directly and instantaneously affecting glycolysis, electron transfer, FAO, Krebs cycle activity, glutamine oxidation, and the urea cycle (Cader et al., 2020;Lowenstein, 1972Lowenstein, , 1990. The lack of technology with temporo-spatial resolution to resolve metabolites across cellular compartments is a particularly acute limitation, compounded by the unparalleled degree of interconnectedness, fast substrate cycles, and redundancies within central purine metabolism. This poses challenges, e.g., for directly measuring flux through IMPDH, and for determining inosine levels at the immunological synapse in situ.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  mice lines; A.K. devised the study and, together with S.S., G.W.S., and K.R. and input from all authors, coordinated the project, designed experiments, interpreted data, and prepared the manuscript.

DECLARATION OF INTERESTS
The University of Cambridge has filed patent applications relating to this work. The authors declare no other competing financial interests.

RESOURCE AVAILABILITY
Lead contact Further information and requests for materials should be directed to and will be fulfilled by the Lead Contact, Arthur Kaser (ak729@ cam.ac.uk).

Materials availability
Unique resources generated in this study are available on reasonable request, although may require completion of a Materials Transfer Agreement.
Data and code availability d RNA Sequencing datasets generated in this study have been deposited at the Gene Expression Omnibus (GSE126473 and GSE147370) and are publicly available as of the date of publication. d This paper does not report original code. d Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon reasonable request. to protein content before addition of 4X laemmli buffer (Bio-Rad) and boiling at 95 C for 5 min. Samples were run on a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo transfer system before blocking for 1 h at room temperature in 5% milk in TBS-T. Membranes were incubated with primary antibodies overnight at 4 C in 5% milk in TBS-T. These were detected by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature and visualized using 20X LumiGlo reagent (Cell Signaling). For measurements of systemic inosine levels plasma was collected by cardiac puncture followed by centrifugation in EDTA-coated tubes for 15 min at 4 C at 2000 g. 20 mL aliquots were taken and prepared for LC-MS by addition of 100 uL 4:1 methanol:water followed by vortexing and centrifugation at 20 000 g. The supernatants were then dried using a centrifugal evaporator (Savant, Thermo-Fisher). Samples were reconstituted in 100 mL ammonium acetate containing 2 mM [ 13 C 10 , 15 N 5 ] adenosine monophosphate and adenosine triphosphate, 10 mM [ 13 C 4 ] succinic acid, a 1 in 5000 diluted [U 13 C, U 15 N] mixture of amino acids (all purchased from Sigma Aldrich) and 50 nM [ 13 C 5 ] inosine (Cambridge Isotope Laboratories) as internal standards. Famin p.254I , Famin p.254V and Famin p.284R mice were fasted for 18 h prior to harvesting.

Extraction of aqueous metabolites
After washing with PBS or 162 mM ammonium acetate adjusted to pH 7.4 (as appropriate), cell pellets were then extracted using the 2:1 chloroform:methanol method described by Folch (Folch et al., 1957) with modifications to the method as previously detailed (Cader et al., 2020). All solvents used were HPLC or LC-MS grade and obtained from Fisher Scientific. Aqueous extracts were stored at À80 C prior to analysis.

LC-MS sample preparation
Aqueous extracts of cells were dried using a centrifugal evaporator (Savant, ThermoFisher) and reconstituted in 10 mM ammonium acetate containing 2 mM [ 13 C 10 , 15 N 5 ] adenosine monophosphate and adenosine triphosphate, 10 mM [ 13 C 4 ] succinic acid, and a 1 in 5000 diluted [U 13 C, U 15 N] mixture of amino acids (all purchased from Sigma Aldrich) as internal standards. Where appropriate, internal standards were omitted during isotopic labeling experiments to prevent contamination with labeled substrates. The samples were then vortexed and sonicated for 5 min, followed by brief pulsed centrifugation to recover maximum volume.
Molecular formula determination using accurate mass and isotopic mass distribution, confirmed by authentic standard, were used to validate identification of inosine as the top-ranking identifiable LC-MS feature of differential abundance between supernatants of Famin +/+ and Famin À/À splenic DCs. For analysis of cell culture supernatants in subsequent experiments, 20 ml of supernatant was aliquoted directly onto a styrene 96 well plate (Corning) followed by dilution with 100 ml of 10 mM ammonium acetate containing 50 nM [ 13 C 5 ] inosine (Omicron Biochemicals) or 50 nM [ 15 N 4 ] inosine (Cambridge Isotope Laboratories) as an internal standard. Where appropriate the internal standard was omitted. For absolute quantitation of both labeled and unlabelled inosine, an inosine calibration line was prepared in the appropriate cell culture medium in the following concentrations: 100 pM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM and 1 mM. These calibrants were then subjected to the same dilution and preparation described above. All plates were sealed with a pre-slit silicone sealing mat prior to injection (Thermo Fisher Scientific).

LC-MS analysis of aqueous metabolites
A Q Exactive Plus orbitrap coupled to a Vanquish Horizon ultra high performance liquid chromatography system was used for all the analysis. LC-MS methodology used corresponds to the ACE C18-PFP and the Phenomenex Gemini-NX protocols described previously (Cader et al., 2020), utilizing identical chromatographic and MS parameters. The majority of analyses (for example detection of nucleotides, nucleosides and organic acids etc.) was carried out using the ACE C18-PFP column and, where appropriate, nucleoside phosphates were measured on a BEH amide HILIC column as detailed in Cader et al. (2020). For analysis of supernatants, where sensitivity was critical, 10 mL was injected with the first minute of chromatography being switched to waste to prevent build-up of matrix containing contaminants in the source of the mass spectrometer.
All solvents and additives used were LC-MS or Optima grade and obtained from Fisher Scientific or Merck.
Hydrazone derivatization of keto acids and hydroxy carboxylic acids in cell culture supernatants and subsequent LC-MS analysis An internal standard solution was prepared by extracting 100 mg of U 13 C lyophilized algae (Sigma) using the Folch extraction described above. Supernatants were dried using a centrifugal evaporator (Savant, Thermo Fisher) and derivatised according to a modified version of the protocol previously described (Han et al., 2013). Briefly, 50 ml of 75% aqueous methanol was added to the dried culture medium followed by 10 ml of the internal standard mix. To this mixture, 30 ml of 250 mM 3-nitrophenylhydrazine (in 50% aqueous methanol), 30 ml of 150 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (in methanol) and 30 ml of 7.5% pyridine (in 75% aqueous methanol) were added sequentially. The resulting mixture was vortexed and samples allowed to derivatise for 1 h on ice. Samples were subsequently quenched with 5 mg/mL butylated hydroxytoluene and 420 ml of water and centrifuged to pellet any salts from the media. The LC gradient employed for the separation of the hydrazone derivatives utilized a binary solvent mixture consisting of mobile phase A, 0.1% formic acid in water and B, 0.1% formic acid in methanol and the column was an Acquity CSH C18 (100 3 2.1mm, 1.7 mm). The gradient program was as follows: 18% B was increased to 90% B in a linear gradient over 6.75 min, held at 90% for a further minute followed by re-equilibration for 1 min to give a total run time of 9 min. The flow rate was 400 ml/min and the column oven temperature was 40 C. The injection volume was 5 ml. To prevent derivatisation reagents from entering the ion source, a switch was employed for the first 2 min of the gradient program. Samples were run in negative ion mode using MS parameters previously described (Cader et al., 2020).

LC-MS data processing
All data were acquired using Xcalibur (Version 4.1, Thermo Fisher Scientific). Targeted processing was carried out using Xcalibur and unbiased analysis using Compound Discoverer (Version 2.1 or Version 3.1, Thermo Fisher Scientific). Untargeted analysis utilized data from both positive and negative ionization modes. Chromatogram peaks for each differential metabolite were manually verified using XCalibur (Version 4.1, Thermo Fisher Scientific) and identities validated using the high-resolution m/z METLIN database (Scripps Research Institute). To confirm identification of inosine and in cases of ambiguity, compound retention times were validated against known external standard solutions. For all cellular and serum metabolite analysis, target peak areas corresponding to metabolites were normalized to total ion content unless otherwise indicated. For absolute quantitation of inosine in supernatants, normalization of target peaks was performed with reference to internal standards, and quantitation performed with reference to a calibration line between 10 pM and 1 mM prepared in the appropriate sample matrix. Relative quantitation of metabolite levels in supernatant tracing studies were not normalized.
All sample data were processed using Compound Discoverer (Version 2.1 and Version 3.1, Thermo Fisher Scientific) to accurately calculate total ion content for use as a normalization factor. For labeling studies, incorporation into specific compounds was determined by accurate mass shift of +1.0034 and +0.9970 for 13 C and 15 N respectively. Endogenous levels of 13 C and 15 N compounds of interest were determined by reference to control samples pulsed with unlabelled compounds of investigation, and endogenous levels subtracted from quantified isotopomers in the labeled samples as applicable.
Determination of extracellular acidification rate and oxygen consumption rate Bone marrow-derived cDC1 stimulated overnight with LPS (O111:B4, Sigma-Aldrich, LPS25), or isolated splenic DCs matured overnight, were seeded prior to analysis at 3 3 10 5 cells per well on Poly-L-lysine-coated plates (Sigma-Aldrich, P8920), as indicated. Cells were then washed twice and incubated for 1 h in XF assay medium (unbuffered DMEM pH 7.4 with 10 mM glucose, 100 mM sodium palmitate and 2 mM L-glutamine) in a non-CO 2 incubator at 37 C as per manufacturer's instructions (Agilent Technologies). Measurements of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined using an XF-96 Extracellular Flux Analyzer (Agilent Technologies). Serial measurements were obtained under basal conditions and following addition of 1 mM oligomycin (Sigma-Aldrich, 75371), 1.5 mM FCCP (Sigma-Aldrich, C2920) and 100 nM rotenone with 1 mM antimycin A (Sigma-Aldrich, R8875 and A867). For determination of glycolysis, ECAR measurements were obtained under basal conditions. Cytoplasmic pH assay Intracellular pH was compared using pHrodo Red AM (Thermo Fisher Scientific, P35372) fluorogenic probe for measurement of cytoplasmic pH according to manufacturer's protocol. In brief, splenic DCs were matured overnight and incubated with 5 mM pHrodo for 30 min at 37 C in a non-CO 2 incubator in Hank's Balanced Salt Solution (HBSS) and washed once before fluorescence signal was measured using a microplate reader (Tecan infinite M1000, Tecan Group or CLARIOstar plus, BMG Labtech) with an excitation/ emission of 560/580nm.
Antigen uptake assay LPS-treated cDC1s or splenic DCs were incubated with 0.05 mg/mL of OVA-AF647 (Thermo Fisher Scientific O34784) in HBSS containing HEPES at 37 C in a non-CO 2 incubator for 30 min, while control cells were kept on ice to account for passive diffusion. Following washes in ice cold HBSS, samples were prepared for analysis by flow cytometry as described above. For analysis of splenic cDC1s and cDC2s cells were gated on CD11c + MHC II + CD8 + CD11b À and CD11c + MHC II + CD11b + CD64 À , respectively.